WO1999029304A1 - Procede de formulation de microspheres a haute teneur en adn surenroule - Google Patents

Procede de formulation de microspheres a haute teneur en adn surenroule Download PDF

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Publication number
WO1999029304A1
WO1999029304A1 PCT/US1998/026462 US9826462W WO9929304A1 WO 1999029304 A1 WO1999029304 A1 WO 1999029304A1 US 9826462 W US9826462 W US 9826462W WO 9929304 A1 WO9929304 A1 WO 9929304A1
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WIPO (PCT)
Prior art keywords
dna
microspheres
emulsion
supercoiled
inhibitor
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Application number
PCT/US1998/026462
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English (en)
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WO1999029304A9 (fr
Inventor
Shuichi Ando
David Putnam
Robert S. Langer
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Massachusetts Institute Of Technology
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Publication of WO1999029304A1 publication Critical patent/WO1999029304A1/fr
Publication of WO1999029304A9 publication Critical patent/WO1999029304A9/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1617—Organic compounds, e.g. phospholipids, fats
    • A61K9/1623—Sugars or sugar alcohols, e.g. lactose; Derivatives thereof; Homeopathic globules
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/1605—Excipients; Inactive ingredients
    • A61K9/1629—Organic macromolecular compounds
    • A61K9/1641—Organic macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyethylene glycol, poloxamers
    • A61K9/1647—Polyesters, e.g. poly(lactide-co-glycolide)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin

Definitions

  • one of the most common techniques for preparation of biodegradable polymer microspheres encapsulating hydrophilic molecules is the double-emulsion solvent evaporation method.
  • the molecule to be encapsulated is placed in aqueous solution while the polymer is dissolved in an organic phase commonly consisting of methylene chloride or ethyl acetate.
  • This primary emulsion is then added to a second aqueous phase and again mixed by homogenization to form the (water-in-oil)-in- water double emulsion.
  • the polymer-containing droplets Upon evaporation of the partially water-miscible solvent, the polymer-containing droplets harden to form microspheres which can then be isolated by filtration or centrifugation. Lyophilization removes water from the interior aqueous phase resulting in a dry suspension of the encapsulated material within the polymer matrix.
  • Lyophilization removes water from the interior aqueous phase resulting in a dry suspension of the encapsulated material within the polymer matrix.
  • the encapsulation efficiency of DNA into the hydrophobic matrix of PLGA was low (-20%) using this method.
  • the use of this method leads to a tendency of plasmid DNA to be converted from its supercoiled state to a nicked or linear state. The preservation of the supercoiled DNA is important because it is known that supercoiled DNA retains the highest level of bioactivity (Xu et al., Biochem. 1996, 35, 5616; Yamaizumi et al., Mol. Cell Biol.
  • DNA therapeutics because of the tendency of DNA therapeutics to degrade during and after the encapsulation process. Specifically, DNA stress induced degradation is encountered during homogenization and lyophilization. Furthermore, the DNA is susceptible to diffusing out of the aqueous phase, thus decreasing the encapsulation efficiency. Therefore, a method of encapsulating DNA based therapeutics that retains the integrity of the DNA (maximizes the supercoiled-DNA content) and increases the encapsulation efficiency would be desirable.
  • the present invention provides methods for the formulation of high supercoiled-DNA content systems and microspheres.
  • the present invention provides a method for the formulation of a high supercoiled DNA content system including formulating an emulsion having a polymer dissolved in organic solvent surrounding an aqueous inner phase containing DNA, and lowering the temperature of the emulsion below the freezing point of the aqueous inner phase.
  • the method includes the step of removing the organic solvent and removing water from the aqueous inner phase to form the system.
  • the system may include microspheres or another implantable structure.
  • the invention provides a method for the formulation of high supercoiled DNA content microspheres which increases the encapsulation efficiency of DNA in microspheres and also prevents the degradation of supercoiled DNA during and after formulation, specifically during the homogenization and lyophilization processes.
  • This method includes the formulation of a primary emulsion, and subsequently lowering of the temperature of the primary emulsion below the freezing point of the aqueous inner phase. Finally, the primary emulsion is transferred to a water-based surfactant solution and subjected to homogenization to form a secondary microsphere emulsion. Stirring of the secondary emulsion allows the removal of the organic phase and hardening of the microspheres, which are then isolated, frozen and lyophilized.
  • a primary emulsion which contains a DNA nicking inhibitor in addition to DNA and buffer.
  • the presence of the DNA nicking inhibitor ensures that the integrity of the DNA is retained.
  • the primary emulsion thus formed with the DNA nicking inhibitor can be utilized in each of the methods described above to provide systems and microspheres with increased encapsulation efficiency and DNA integrity.
  • the present invention provides a method for the cryopreparation of water soluble low molecular weight compounds to increase their encapsulation efficiency.
  • Figure 1 represents a summary of the microsphere manufacturing procedure
  • Figure 2 depicts the effect of homogenization rate on the diameter of microspheres
  • Figure 3 depicts agarose gel electrophoresis of DNA in microspheres
  • Figure 4 depicts the effect of excipients on retaining supercoiled DNA under microsphere procedure such as homogenization and lyophilization
  • Figure 5 depicts the effect of homogenization rate, cryopreparation and addition of EDTA on remaining supercoiled DNA
  • Figure 6 depicts the effect of excipients on retaining supercoiled DNA
  • Figure 7 depicts the effect of lyophilization on retaining supercoiled DNA
  • Figure 8 depicts the effect of saccharides on DNA stability during lyophilization
  • Figure 9 depicts agarose gel electrophoresis of DNA incubated at room temperature in PBS at pH 7.4
  • FIG. 10 depicts the analytical methods employed for DNA structure analysis
  • the present invention provides methods for the formation of high supercoiled DNA content systems and microspheres.
  • the present invention utilizes a cryopreparation method in which a primary emulsion having an aqueous inner phase and a surrounding organic phase is first formed, and subsequently the temperature of the primary emulsion is lowered below the freezing point of the aqueous inner phase.
  • the present invention also provides a method for the formulation a high supercoiled DNA content system in which, in addition to the cryopreparation method described above, the organic solvent from the surrounding organic phase is removed and the water is removed from the aqueous inner phase to formulate the high supercoiled DNA content system.
  • the inventive method also provides for the formulation of high supercoiled DNA content micrspheres by formulating a primary emulsion comprising a polymer dissolved in organic solvent surrounding an aqueous inner phase containing DNA, lowering the temperature of the primary emulsion below the freezing point of the aqueous inner phase, forming a secondary microsphere emulsion and forming the DNA content microspheres.
  • the present invention also provides, for each of the methods described above, the use of a DNA nicking inhibitor in addition to DNA and buffer in the formation of the primary emulsion.
  • a DNA nicking inhibitor is particularly preferred for each of the methods described above because the presence of the DNA nicking inhibitor ensures that the integrity of the DNA is retained.
  • the encapsulation efficiency of water soluble low molecular weight compounds such as peptides or hormones is increased by the cryopreparation step employed after formation of the primary emulsion.
  • the cryopreparation step decreases the ability of the water soluble low molecular weight compound to diffuse out of the aqueous phase.
  • the cryopreparation step for water soluble molecular weight compounds is performed similarly to that for the cryopreparation step for DNA as described below.
  • the method of the invention is described with reference to the flow diagram of Figure 1, which shows the microsphere manufacturing procedure.
  • the primary emulsion is prepared according to standard methods and consists of a polymer dissolved in an organic solvent and surrounds an aqueous inner phase containing DNA and a DNA nicking inhibitor.
  • poly(lactic-co-glycolic)acid is the polymer used and the organic solvent is methylene chloride. It will be appreciated by those of ordinary skill in the art that other polymers and organic solvents may be used according to the method of the presently claimed invention.
  • wall-forming materials include, but are not limited to, poly(lactide), poly(glycolide), poly(caprolactone), and poly(hydroxybutyrate).
  • solvents may be chosen by one of ordinary skill in the art with the limitation that the solvent must dissolve the wall material and also that the solvent have limited solubility in the extraction medium.
  • a preferred solvent is ethyl acetate.
  • a DNA nicking inhibitor is employed in the presence of a chelator such as EDTA or DTPA. A suitable concentration of EDTA is above 0.5 mM. In another embodiment of the invention, the DNA nicking inhibitor is employed alone.
  • Preferred DNA nicking inhibitors to be used in the claimed invention include carbohydrates, disaccharides, higher molecular weight saccharides, or water soluble polymers.
  • Specific carbohydrates include, but are not limited to, fructose, erythrose, threose, ribose, arabinose, xylose, lyxose, allose, altrose, glucose, mannose, gluose, idose, galactose, erythrulose, ribulose, xylulose, psicose, fructose, sorbose, tagatose, fructofuranose, ribofuranose, ribose, deoxyribose, mannitol, and sialic acid.
  • Specific disaccharides include but are not limited to sucrose, lactose, maltose, cellobiose, trehalose, and lactulose.
  • Specific polysaccharides include but are not limited to starch, glycogen, cellulose, chondroitin, keratin, haparin, dermatan, and haluronic acid.
  • Specific water soluble polymers include but are not limited to polyethylene oxide and polyethylene glycol.
  • the concentration of the DNA nicking inhibitors is preferably in the range of 100-300 mM, and most preferably about 300 mM. Specifically, concentrations of more than 100 mM can be utilized in the present invention, with the limitation that the saccharide must still be soluble.
  • the primary emulsion temperature is then lowered below the freezing point of the aqueous inner phase.
  • the temperature of the primary emulsion is lowered by submersion into liquid nitrogen.
  • Other methods of lowering the temperature of the primary emulsion include immersion of the primary emulsion (contained in a sealed vessel) into a solution of dry ice and acetone.
  • the organic phase is then melted until the suspension reaches -30°C. Temperatures in the range of -50 °C to -10°C may also be employed in this step.
  • the water-based surfactant solution is a solution of 5% polyvinyl alcohol containing 300 mM lactose. It will be appreciated by those of ordinary skill in the art that other water based surfactant solutions include but are not limited to carboxymethyl cellulose, gelatin, poly(vinylpyrrolidone), Tween 80, and Tween 20.
  • the secondary emulsion is formed using homogenization preferably at a range of 5,000 to
  • the homogenization occurs most preferably at about 7000 RPM. It will be appreciated by one of ordinary skill in the art that the desired size of the spheres depends upon the rate of the homogenization. Figure 2 shows the effect of the homogenization rate on the diameter of the microspheres. For the purposes of the present invention, spheres with a size of approximately 5 microns are desired, because of the ability of microspheres of this size to enter into phagocytic cells and deliver the therapeutic agent.
  • the microspheres are formed in one embodiment by adding the secondary emulsion to a surfactant solution such as polyvinyl alcohol (most preferably 1%) in 300 mM lactose, and stirred for at least 3 hours at room temperature to remove the methylene chloride.
  • a surfactant solution such as polyvinyl alcohol (most preferably 1%) in 300 mM lactose, and stirred for at least 3 hours at room temperature to remove the methylene chloride.
  • microspheres are collected by centrifugation, washed with distilled water and lyophilized for 15 hours.
  • the collected microspheres described above preferably have mean diameters of less than 1 mm, and more preferably less than 10 microns, and most preferably about 4.8 microns. Additionally, the microspheres contain most preferably greater than 88% of the supercoiled DNA ( Figures 3 and 4). Moreover, the encapsulation efficiency is most preferably about 89%. For the purposes of the present invention encapsulation efficiency is determined by comparing the amount of DNA initially used with the amount of DNA actually encapsulated.
  • the resulting microspheres had a mean volume diameter of 4.5 ⁇ m, a remaining supercoiled-DNA content of 39%, and a DNA encapsulation efficiency of 23%. It is apparent that cryopreparation prevents degradation of DNA and increases the encapsulation of DNA.
  • DNA encapsulation efficiency is caused by preventing its diffusion out of the inner aqueous phase by freezing the primary emulsion. Furthermore, addition of DNA- nicking inhibitors to the DNA solution is important to prevent DNA degradation during this microsphere manufacturing process. Ninety-five percent of supercoiled DNA was retained before lyophilization and 88% of supercoiled DNA was retained after lyophilization as shown in Figure 3. In addition, Figure 4 compares the supercoiled DNA content in microspheres using water, EDTA, PBS, or lactose in the DNA solution.
  • Figure 6 indicates that DNA degradation during cryopreparation was not inhibited by the addition of PBS (1 mM K 2 HPO 4 , 10 mM Na 2 HPO 4 , 137 mM NaCl, 2.1 mM KC1 pH 7.0), Tris, or lactose to the DNA solution.
  • PBS 1 mM K 2 HPO 4 , 10 mM Na 2 HPO 4 , 137 mM NaCl, 2.1 mM KC1 pH 7.0
  • the supercoiled-DNA content was increased from 75% to 95%.
  • the exact mechanism of DNA stabilization is unknown. However, it is apparent that the presence of both lactose and EDTA in the DNA solution is important for the stabilization of supercoiled DNA against degradation during cryopreparation. Stability of DNA Structure during Lyophilization:
  • Plasmid DNA (pCMV- ⁇ -gal) was purified from E.coli (DH5 ⁇ ) using Plasmid Mega Kit column isolation (QIAGEN, CA), followed by ethanol precipitation.
  • Example 1 Crypreparation: DNA containing microspheres were prepared using a cryopreparation method based on the water-in-oil-in-water double emulsion solvent- evaporation method. The two phases, consisting of 250 ⁇ m of DNA solution (250 ⁇ g of DNA) and 7 mL of methylene chloride containing 200 mg of PLGA, were emulsified by sonication for 10 s (ultrasonic probe, Sonic & Materials, Inc.) At room temperature.
  • the primary emulsion temperature was then lowered below the freezing point of the aqueous inner phase by liquid nitrogen immersion, and 50 mL of a 5% PVA solution (4-7 °C) was added and homogenized at 5000-9000 rpm for 14 s (Silverson L4R homogenizer). After homogenization, the resulting emulsion was diluted in 100 mL of 1% PVA, and the system was stirred magnetically for 3 h to allow for evaporation of the organic solvent. Microspheres were finally collected by centrifugation and washed 3 times with water to remove excess PVA. Note that all of the PVA solutions were adjusted to the osmotic pressure of the inner aqueous phase using agents such as saccharides. The microspheres were resuspended in approximately 1 mL of water, frozen in liquid nitrogen, and lyophilized at room temperature for 24 h on a Labconco Freeze-Dryer 8.
  • Example 2 Achieving DNA Stability against Lyophilization Using Excipients: The effect of lyophilization on DNA was studied by directly lyophilizing DNA samples
  • Example 3 Optimized Microsphere Preparation: The two phases, consisting of 250 ⁇ L of DNA in water (750 ⁇ g of DNA) containing 1 mM EDTA and 300 mM lactose
  • DNA was compared toa standard curve, using plasmid DNA, which was linear from 1 to 50 ng/mL.
  • Example 6 Particle Size of Microsphere: Particle size distribution of microspheres was analyzed by a Coulter Multisizer II (Coullter Electronics Inc., Hialeah, FL), and the mean volume diameter distribution was determined.
  • Coulter Multisizer II Coullter Electronics Inc., Hialeah, FL

Abstract

L'invention concerne un procédé de formulation de microsphères à haute teneur en AD surenroulé. On forme une émulsion primaire renfermant éventuellement, en plus d'ADN pourvu ou dépourvu de tampon, un inhibiteur de coupure d'ADN. La température de l'émulsion primaire est abaissée au-dessous du point de congélation de la phase aqueuse intérieure, ce qui permet d'obtenir un rendement d'encapsulation accru par réduction de la vitesse diffusion de l'ADN hors phase aqueuse. Ensuite, on transvase l'émulsion primaire dans une solution de tensio-actifs à base d'eau et on la soumet à une homogénéisation de manière à former une émulsion de microsphères secondaire. On retire la phase organique et on laisse durcir les microsphères avant de les isoler, de les congeler et de les lyophiliser.
PCT/US1998/026462 1997-12-12 1998-12-11 Procede de formulation de microspheres a haute teneur en adn surenroule WO1999029304A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US6935897P 1997-12-12 1997-12-12
US60/069,358 1997-12-12
US09/209,032 US6197229B1 (en) 1997-12-12 1998-12-10 Method for high supercoiled DNA content microspheres
US09/209,032 1998-12-10

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WO1999029304A1 true WO1999029304A1 (fr) 1999-06-17
WO1999029304A9 WO1999029304A9 (fr) 1999-09-23

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6572894B2 (en) 1995-11-24 2003-06-03 Actipac Biosystems Gmbh Process for the production of morphologically uniform microcapsules and microcapsules that are produced according to this process
EP1098667A1 (fr) * 1998-07-17 2001-05-16 Mirus Corporation Systemes chelateurs servant a acheminer des composes jusqu'aux cellules
EP1098667A4 (fr) * 1998-07-17 2002-09-11 Mirus Corp Systemes chelateurs servant a acheminer des composes jusqu'aux cellules
WO2002003961A1 (fr) * 2000-07-07 2002-01-17 Corixa Corporation Microspheres et adjuvants utilises pour l'administration de vaccins a base d'adn
US6899898B2 (en) 2000-12-21 2005-05-31 Nektar Therapeutics Induced phase transition method for the production of microparticles containing hydrophobic active agents
US7252842B2 (en) 2000-12-21 2007-08-07 Alrise Biosystems Gmbh Induced phase transition method for the production of microparticles containing hydrophilic active agents
CN107874257A (zh) * 2017-11-09 2018-04-06 大连工业大学 高包埋率小粒径南极磷虾油纳米粒的制备方法
CN107874257B (zh) * 2017-11-09 2021-05-07 大连工业大学 高包埋率小粒径南极磷虾油纳米粒的制备方法

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